PROJECT SUMMARY (PROJECT 2) The focus of this project is to exploit the Drosophila model system to study the role of retrotransposable elements (RTEs) in the progression of cellular dysfunction that occurs during aging, and to develop inter- ventions that suppress RTE activity and extend healthy life span. The maintenance of repressive hetero- chromatin declines with age, resulting in increased expression and mobilization of RTEs across species as diverse as fruitflies and mice. We hypothesize that the increase in the expression and mobilization of RTEs leads to an accumulation of damage to the genome of somatic cells, causing loss of cellular and organismal homeostasis, and thus promoting aging. We will use the powerful molecular and genetic tools and the short life span of Drosophila to determine the effects of RTEs on aging, and in particular, we will do this over the entire life span of the organism, something that would not be possible in humans, and would be prohibitively time- consuming and expensive in any mammalian model system on a large scale. Confirmation of a mechanistic relationship between RTE activity and aging will provide the groundwork for developing interventions that diminish RTE activity and genomic damage during aging, and should extend healthy life span. The aims of this proposal are to test the hypotheses that: (i) loss of chromatin-regulated suppression of RTE activity with age is associated with increased DNA damage and genome instability; (ii) decreases in sirtuins with age lead to elevated RTE activity that can be reversed by increasing sirtuin activity; and (iii) interventions that suppress RTE activity in somatic cells can extend healthy life span. To better understand the relationship between RTE activity, genomic damage and longevity, as well as to develop new interventions that can extend healthy life span, in Aim 1 we will develop high throughput DNA sequencing methods (collaboration with Project 1, Core B), deploy available and new RTE reporters (collaboration with Core B) to measure mobilization of RTEs in aging Drosophila, and carry out a forward genetic screens to identify new genes and pathways involved in the suppression of RTEs. We will examine the effects of (i) age, (ii) dietary restriction (DR), and (iii) genetic interventions that stabilize heterochromatin, such as increasing Su(var)3-9, HP1a, Dicer-2, Piwi or decreasing Adar expression, on RTE activity and mobilization. In Aim 2, to determine the role of Sir2 and Sirt6 in the repression of RTE activity during aging, we will combine the tools developed and utilized in Aim 1 to monitor RTE activity with genetic manipulations that increase or decrease the expression of Sir2 and of Sirt6 activity (collaboration with Project 3 and Core B). In Aim 3, we will (i) determine the effects of the genetic interventions developed in Aims 1 and 2 on the life span of flies (collaboration with Projects 1 and 3 and Core B), and (ii) test the effects of nucleoside reverse transcriptase inhibitors (nRTIs) and RNAi knockdowns of specific retrotransposons on RTE mobilization and lifespan (collaboration with Projects 1 and 3 and Core B).